TRPV1 MODULATORY GENE PRODUCT THAT AFFECTS TRPV1-SPECIFIC PAIN BEHAVIORAL RESPONSES IDENTIFIED IN A FUNCTIONAL SCREEN OF AN HSV-BASED cDNA LIBRARY

ABSTRACT

The invention provides a method for ameliorating chronic pain signaling involving transient receptor potential cation channel subfamily V member 1 (TRPV1) by expressing PP1α in neurons. The invention also provides HSV vectors for expressing PP1α within neurons and compositions comprising such vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of co-pending U.S. patentapplication Ser. No. 15/442,521, filed Feb. 24, 2017, which is acontinuation of U.S. patent application Ser. No. 14/690,038, now U.S.Pat. No. 9,580,699, which was filed Apr. 17, 2015 and which claims thebenefit of U.S. Provisional Patent Application 61/980,925, filed Apr.17, 2014. The entire contents of these prior applications areincorporated herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant NumberDK044935 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

Chronic pain represents a major cause of morbidity, significantlyimpairing quality of life and imposing a substantial financial andhealthcare burden. The wide distribution of a limited range ofneurotransmitters, receptors, and ion channels in the nervous systemmakes it difficult to selectively target pain-related pathways usingdrugs that are administered systemically. As a result, tolerance, abuse,and deleterious side-effects limit the use of all currently availabletherapeutics.

We have previously used replication-defective HSV-based gene therapyvectors to deliver inhibitory neurotransmitters or gene products thatattenuate the action of pro-nociceptive molecules to primary neuronalafferents of the peripheral nervous system (PNS), thereby blockingnociceptive neurotransmission in a variety of pre-clinical pain models[Goss et al., 2001; Goss et al., 2002; Goss et al., 2011; Hao et al.,2006; Hao et al., 2009; Miyazoto et al., 2010; Srinivasan et al., 2008;Wilson et al., 1999; Yokoyama et al., 2009] and more recently inpatients [Fink et al., 2011)].

The transient receptor potential cation channel subfamily V member 1(TRPV1) is one of 28 members of the transient receptor potential (TRP)non-selective cation channel superfamily and an important regulator ofprimary afferent nociceptive activity and pain signaling [Caterina &Julius, 2001]. TRPV1 can be induced by binding of the agonist capsaicin,as well as by protons and temperatures above 42° C. [Caterina et al.,1997; Tominaga et al., 1998]. TRPV1 has been documented to contribute tothe chronic pain state in patients with arthritis, cancer, cystitis,diabetic neuropathy and post-herpetic neuralgia [Brederson et al., 2013;Bohlen & Julius, 2012; Roberson et al., 2011]. TRPV1 is primarilylocalized to the surface of small unmyelinated C-fibers that are thoughtto be the primary nociceptors as TRPV1 levels have been associated withthermal hyperalgesia (TH) in inflammatory and neuropathic pain models[for review see Winter et al., 2013].

Over the last decade, a number of gene products have been identifiedthat modulate TRPV1 activity via phosphorylation of residues within thecytoplasmic domains of TRPV1 that assist in sensitization of thereceptor. These products include calcium/calmodulin-dependent kinase(CamKinase-II) [Price et al., 2005; Zhang et al., 2011], protein kinaseA (PKA) [Lee et al., 2012; Suguira et al., 2004: Varga et al., 2006] andthe epsilon isoform of protein kinase C (PKCe) [Hucho et al., 2012].TRPV1 activity is also modulated in the opposite manner (i.e.desensitization) via dephosphorylation of residues located in thecytosolic domains of the receptor. An established example of adesensitizing molecule is calcineurin, a Ca²⁺-calmodulin-dependentserine/threonine protein phosphatase, also known as Protein Phosphatase2B (PP2B) [Chaudhury et al., 2011; Jeske et al., 2006; Mohapatra & Nau,2005; Por et al., 2010].

BRIEF SUMMARY OF THE INVENTION

The invention provides for the use of a novel pain inhibitor gene andcDNA encoding a product, PP1α, that is specific to TRPV1 as a potentialpain gene therapy. The PP1α cDNA was identified in a screen of a cDNAlibrary derived from PC12 cells differentiated with NGF and expressedfrom a replication-competent HSV vector that also expresses two copiesof the TRPV1 gene.

When expressed from replication-defective HSV, the PP1α product of theidentified cDNA can target nocifensive behaviors associated with TRPV1but not TRPM8 or TRPA1, attesting to the specificity of this geneproduct. The TRPV1 specificity of PP1α is novel and crucial for a genetherapy approach to treating chronic pain as both TRPM8 and TRPA1 areinvolved in a variety of important responses including touch, cold andother sensations that are preferably left unaltered by the treatment.Thus, using a gene product like PP1α that specifically targetsTRPV1-activated pain provides a layer of safety by decreasing unwantedside effects. For example, patients treated with the PP1α gene therapyapproach will still be able to actively sense cold stimuli so that theycan respond to a cold stimulus that would result in acute pain.

We have expressed PP1α from a replication-defective HSV vector that isdeleted for the essential ICP4 and ICP27 genes. We have shown thatHSV-based expression of PP1a, similar to that of a dominant-negativeversion of TRPV1 referred to as Poreless (PL), can reduce TRPV1sensitization by the agonist capsaicin in rat primary DRG neurons invitro as measured by calcium imaging and capsaicin-induced thermal painresponses in vivo following vector injection into the footpads ofSprague-Dawley male rats. In addition, we have shown that HSVvector-expressed PP1α did not alter the function of other TRP channelssuch as TRPM8 following menthol or icilin induction in the cold ramptest, or TRPA1 in the formalin pain test.

PP1α is the first and only product tested preclinically as a genetherapy for pain that specifically targets TRPV1-associated pain anddoes not affect the normal TRPM8 and TRPA1 responses.

The PP1α replication-defective HSV vector can also ameliorate bladderpain in rats in which another TRPV1 agonist, Resiniferatoxin (RTx), isused to activate the pain response. PP1α gene therapy may also interferewith other types of pain such as arthritis pain, VZV-associatedPost-Herpetic Neuralgia (PHN) pain, bone cancer pain, the spinal nerveligation (SNL) or chronic constriction injury (CCI) models ofneuropathic pain, and complete Freund's adjuvant (CFA)-inducedinflammatory pain.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A-1C schematically depict HSV vectors and cDNA library screeningprotocol for TRPV1 inhibitors. (A) Virus vectors. HSV KOS-BAC containsthe BAC sequences that contain the E. coli origin and a chloramphenicolresistance gene for selection in bacteria. TTA BAC contains: two copiesof the TRPV1 gene, under control of the TK early gene promoter that willbe active when the virus replicates, were inserted into the UL23 andUL41 gene loci within the KOS-BAC genome; the Gateway sequences wereinserted into the joint region; the PC12 cell cDNA library wasintroduced into the gateway cassette under the control of the HCMVpromoter that will allow robust expression of the genes within thelibrary prior to expression of TRPV1. (B) The library was generated fromdifferentiated and non-differentiated PC-12 cells (representing genesranging in size from ˜0.5-3 Kb) was inserted into the TTA BAC via aGateway recombination LR clonase reaction. (C) The TTA library wasscreened (˜500 K PFU) by infecting Vero cells followed by the additionof 0.5 μM of the TRPV1 agonist capsaicin. Only viruses making a productthat counteracts the capsaicin induction of TRPV1 will allow virus toreplicate and survive. Following a second screen 25/46 isolatesremained. cDNAs were made of the 25 candidates, re-introduced into theTTA BAC and re-screened in the presence of capsaicin. The lone remainingisolate from the final screen was sequenced and showed direct homologyto the gene product protein phosphatase 1-α or PP1α.

FIG. 2 presents data verifying the ability of the PP1α cDNA to enablevirus replication in the presence of capsaicin. In order to verify thatthe PP1α isolate was working and show that its actions were throughdown-regulating capsaicin activation of the TRPV1 receptor, we comparedthe growth of the PP1α isolate with the TTA BAC that lacks the cDNAlibrary (negative control), and to a positive control virus thatcontains a known modulator of TRPV1 called Poreless (PL), adominant-negative mutant form of TRPV1 that when expressed poisons theaction of the wild-type TRPV1. We had previously used the Poreless-TRPV1expressing virus to verify that our capsaicin screening procedure wasvalid in comparison to the TTA BAC virus. Cells infected with the 3viruses (PP1α, TTA and PL) were either left untreated to show maximumlevels of virus replication (black bars), or treated with 0.5 μMcapsaicin (grey bars). The TTA negative control virus replicated in theabsence of capsaicin but no virus was detected when 0.5 μM capsaicin wasadded, as seen during the screening process. The PL positive controlvirus in the presence of capsaicin was able to replicate to about 50% ofthe levels seen without capsaicin similar to that observed when we firsttested the entire screening process. Finally, the PP1α virus showedsimilar levels of replication to that of the PL positive control virusin the presence of 0.5 μM capsaicin, verifying what we had observed inthe screening process.

FIG. 3 presents data demonstrating the ability of HSV vector expressedPP1α to alter TRPV1 activity in fetal dorsal root ganglia neurons usingcalcium imaging. Fetal DRG neurons were isolated from day e15 ratembryos and cultured in Neurobasal Medium supplemented with B27 and NGF.At 1-2 weeks post-plating, cultures were infected with vectorreplication-defective HSV vectors expressing GFP (negative control),poreless (PL) positive control, or the PP1α test vector. Calcium imagingwas assessed at 3 hours post-infection in the presence of 0.5 μMcapsaicin using Fura-2, a cell-permeable fluorescent Ca²⁺ indicator tomeasure the influx of Ca²⁺. The data in the bar graph depicts the Fura-2Ratio (F340/F380) which is proportional to the concentration ofintracellular calcium present within the DRG neurons. The resultsrepresent the average of multiple measurements from DRG neurons infectedwith the three viruses. Both poreless and PP1α expression resulted instatistically significant reductions in the levels of intracellularcalcium, that supports their augmentation of the capsaicin induction ofthe TRPV1 channels.

FIGS. 4A-4B present data demonstrating the inhibition of chronic painsignaling using a vector expressing PP1α protein or Poreless TRPV1. MaleSprague-Dawley rats were injected subcutaneously into the right rearhind paw with 1×10⁸ pfu of either a control vector (vHG, N=6) or avector expressing a poreless TRPV1 (PL, N=6) or with 1×10⁷ pfu of eithera control vector (GFP, N=6) or PP1α-expressing vector (N=6). One, twoand three weeks later, thermal response was assessed by placing eachanimal into a plexiglass enclosure on a glass surface maintained at 30°C. (Hargreaves apparatus). After a 15 min accommodation period, a lightbeam was focused onto the mid-plantar area of each hind paw and theamount of time it took each animal to move its paw from the heat sourcemeasured. The average of three trials per hind paw for each animal wasused to determine each animal's thermal response. Values represent theratio of response between the ipsilateral (injected) and contralateral(non-injected) paw. SPSS stats package determined one-way ANOVA *p<0.01.

FIGS. 5A-5B present data demonstrating the inhibition of capsaicininduced thermal allodynia in rats by footpad injection ofreplication-defective vectors expressing Poreless TRPV1 or PP1α. MaleSprague-Dawley rats were injected subcutaneously into the right rearhind paw with 1×10⁸ pfu of either a control vector (GFP, N=6) or theporeless TRPV1 (PL, N=6) or with 1×10⁷ pfu of either a control vector(GFP, N=6) or PP1α-expressing vector (PP1α, N=6). One week later,capsaicin (10 mM in 50% ethanol) was applied to the plantar surface ofthe right hind paw to lower the thermal pain threshold. Animals wereplaced on a dynamic hot place where temperature increased from 30° C. to45° C. over a 15 minute period. During each degree interval the totalnumber of pain-related behaviors (licking, paw withdrawals) for theinjected ipsilateral paw were counted and plotted as the number ofresponses. Both PL and PP1α alter the thermal allodynia behavioralresponse to capsaicin.

FIGS. 6A-6B present data demonstrating that Poreless TRPV1 and PP1α HSVvectors have no effect on cold-induced pain. Since noxious coldtemperature is transduced primarily by TRPM8 receptors, our porelessTRPV1 vector should not interfere with its function. Male Sprague-Dawleyrats were injected subcutaneously into the right rear hindpaw with 1×10⁸pfu of either a control vector (GFP, N=6) or the poreless TRPV1 (PL,N=6) or with 1×10⁷ pfu of either a control vector (GFP, N=6) orPP1α-expressing vector (PP1α, N=6). 7-10 days later, animals were placedon a dynamic cold plate where temperature decreased from 30° C. to 4° C.over a 15 minute period. Additionally, with the PP1α vs. GFP control, asolution of 5% menthol was placed on the right hindpaw prior to placingthe rat on the cold ramp. During each degree interval the total numberof pain-related behaviors (licking, paw withdrawals) for the injectedipsilateral and non-injected contralateral were counted and the totalnumber of licks and withdrawals for the injected hindpaw are plotted.Neither PL or PP1α vectors had any effect on the cold-related behavioralresponses in the rats compared to the GFP control-injected rats even inpresence of TRPM8 agonist menthol.

FIG. 7 presents data demonstrating that HSV vector-mediated PP1αexpression has no effect on TRPA1 nociception behavior evaluated usingthe formalin footpad test. Male Sprague-Dawley rats were injectedsubcutaneously into the right rear hindpaw with 1×10⁷ pfu of either acontrol vector (GFP, N-6) or PP1α-expressing vector (PP1α, N=6). 7-10days later, a dilute solution of formalin (2.5%) was injectedsubcutaneously in the plantar aspect of the same foot that had receivedthe viral inoculation, and the rats then placed into a 48-27-20 cmplastic box positioned over a mirror tilted at a 45° angle. Beginning 30s after the injection of formalin, and once every 10 min thereafter,nocifensive behaviors were recorded by a blinded observer for 3 min. Aweighted pain score was derived based on the amount of time the animalexhibited each behavior during the 3-min period of observation[0=plantar surface of foot flat against surface; 1=foot cupped with onlytoes touching surface; 2=lifting of foot from surface; 3=completewithdrawal of foot with licking]. The weighted pain score was plottedagainst time, and compared between control and treatment groups using aone-way ANOVA.

FIGS. 8A-8B present data demonstrating that PP1α reduces both thermaland mechanical nocifensive behavioral responses in the rat model ofVZV-induced PHN. Baseline mechanical allodynia (MA) and thermalhyperalgesia (TH) nocifensive responses were measured in ten maleSprague Dawley rats using the von Frey hair up-down method (MA) or theHargreaves (TH) apparatus. Rats were then injected into their righthindpaws with MeWo infected with 10⁵ pfu VZV strain pOka (parent of Oka,similar to VZV vOka vaccine strain). At 7 and 14 days post VZV, MA andTH behaviors were measured. Next, on DAY-0 the rats were injected intothe same hindpaws with 10⁷ pfu of either the control GFP-expressingreplication-defective HSV vector or the PP1α-expressing vector. AtDAY-7, rat MA and TH behaviors were assessed. All behavioral scores areplotted as the average response ratio for the ipsilateral injectedhindpaw to the contralateral un-injected hindpaw. VZV-PHN mechanical andthermal nocifensive behaviors were observed at Day 0, however, only thecontrol GFP-expressing animals retained these responses at Day 7 whilethe PP1α-expressing vector dramatically reduced both MA and THnocifensive behaviors in a statistically significant manner. The averagepaw withdrawal latency time for the uninjected non-inured rats rangedfrom 7-10 seconds while those for the VZV-injected/PP1α vector injectedrats ranged from 13-25.

FIGS. 9A-9C present data demonstrating that PP1α reduces both thermaland mechanical nocifensive behavioral responses in the rat model ofVZV-induced PHN over 6 weeks post injection of PP1α HSV vector comparedto control GFP vector. Baseline MA and TH nocifensive responses weremeasured in ten male Sprague Dawley rats. Rats were then injected intotheir right hindpaws with MeWo cells infected with 10⁵ pfu of VZV strainpOka. At 7 and 14 days post VZV, MA and TH behaviors were measured whereall rats displayed pain. Next, on DAY-15 the rats were injected into thesame hindpaws with 10⁷ pfu of either the control GFP-expressing HSVvector or the PP1α-expressing vector. At weekly intervals, rat MA and THbehaviors were assessed. All behavioral scores are plotted as theaverage response ratio for the ipsilateral injected hindpaw to thecontralateral un-injected hindpaw. VZV-PHN mechanical and thermalnocifensive behaviors were observed by 14 days post VZV pOka. However,only the rats injected with the control GFP-expressing vector retainedthese responses (7 days post HSV vector injection and later) while thePP1α-expressing vector dramatically reduced both MA and TH nocifensivebehaviors in a statistically significant manner. Even at 6 weeksfollowing HSV vector injection, the PP1α vector continues to block thepainful behaviors in the PHN rats at statistically significant levels.

FIG. 10 presents data demonstrating Inhibition of CFA-inducednocifensive behavior using an HSV vector expressing PP1α andPoreless-TRPV1. Male Sprague-Dawley rats (200-250 g) were assessed forbaseline thermal behavior by placing each rat into a plexiglassenclosure on a glass surface maintained at 30° C. (Hargreavesapparatus). After a 15 min accommodation period, a light beam wasfocused onto the midplantar area of each hind paw and the amount of timeit took in seconds for each animal to move its paw from the heat sourcewas measured. The average of three trials per hind paw for each animalwas used to determine each animal's thermal response. Rats were theninjected subcutaneously into the right rear hind-paw with 1×10⁸ pfu ofeither a control vector (DAP-GFP, N=4), a vector expressing a PP1α(DAP-PP1α, N=4), or vector expressing a Poreless-TRPV1 (DAP-PL, N=4).Three weeks later, thermal response was again assessed and the averageand standard deviation of the thermal response times (in seconds) shownfor the injected right hind-paw as time point 0 days post CFA injection.After this Day 0 measurement, 100 μL of complete Freund's adjuvant (CFA)was injected into the right hind-paw as previously described (Goss etal., 2011) and the thermal response was again assessed at 1, 2 and 3days post-CFA injection.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention provides a herpes simplex virus (HSV)vector comprising an expression cassette, which comprises a nucleic acidsequence encoding PP1α. The vector backbone is preferably replicationincompetent in vivo. For example, the HSV vector can be engineered tocontain deletions and/or inactivating mutations of genes essential forviral replication in cells not otherwise complementing for such viralproteins. For example, a suitable HSV vector backbone can lackfunctioning ICP4 and ICP27 genes, as well as other genes (e.g., ICP0).Additionally, the HSV vector can be targeted to particular types ofcells, such as neurons that are involved in the sensation of pain (e.g.,C-fibers). Examples of replication-deficient and targeted HSV vectors,and their methods of production and replication are known in the art.See, e.g., U.S. Pat. Nos. 8,309,349, 8,003,622, 7,825,231, 7,531,167,7,078,029, 6,261,552, 5,998,174, 5,879,934, 5,849,572, 5,849,571,5,804,413, and 5,658,724; US patent application publication numbers20020090382, 20070178069, 20070207124, 20080289058, 20090238807, and20110213017, and international patent application publication numbersWO/2011/130749, WO/2008/143875, WO/2006/050211, WO/1999/061067, andWO/1999/006583, each of which is incorporated herein by reference.

Within the vector, preferably the nucleic acid sequence encoding PP1α isoperably linked to a promoter sequence other than the native PP1αpromoter. For example, the nucleic acid sequence encoding PP1α can beoperably linked to a strong constitutive promoter, such as the hCMV1major immediate-early (IE) gene promoter. For expression within neurons,it will be appreciated that the nucleic acid sequence encoding PP1α canbe operably linked to a neuronal-specific promoter. For example, sincePP1α inhibits TRPV1, within the inventive vector, the nucleic acidsequence encoding PP1α can be operably linked to the TRPV1 promoter,which will aid in confining expression of PP1α to cells which alsoexpress TRPV1. Alternatively, the nucleic acid sequence encoding PP1αcan be operably linked to a non-neuronal promoter that provideslong-lasting gene expression within neurons, such as the ubiquitin (Ub)promoter.

For use in the context of the present invention, preferably (a) thePP1α, (b) the genetic regulatory elements to which it is operably linked(e.g., its promoter) within the vector, and more preferably (c) both thePP1α and its genetic regulatory elements within the vector, areco-specific with the species of cells to which the vector isadministered. Thus, for medical use in humans, preferably the PP1α, itspromoter within the vector, and more preferably both, are humansequences.

It will be appreciated that the inventive HSV vectors can be propagatedinto a stock comprising numerous such vectors. Thus, the inventionprovides a stock comprising such vectors, which can have any suitabletiter of vectors. Desirably, the stock has a titer of between about 10⁸and about 10¹¹ pfu/ml or greater.

The invention also provides a pharmaceutical composition comprising thevectors containing the nucleic acid sequence encoding PP1α as describedherein, and a pharmaceutically-acceptable carrier. Any suitable carriercan be used, so long as it is compatible with the HSV vectors and alsosuitable for pharmaceutical use. Typically, such carriers arephysiological saline solutions, which facilitate administration via skinprick, or via subdermal, intramuscular, or parenteral injection.However, other carriers (e.g., salves, creams, patches, and the like fortransdermal administration) also can be used. For treating CNSconditions, carriers suitable for intracranial administration can beemployed.

The invention also provides a method of treating pain within a mammal inneed thereof. The method comprises administering a pharmaceuticalcomposition comprising the vectors comprising an expression cassette,which comprises a nucleic acid sequence encoding PP1α, to the mammal inan amount and at a location sufficient to result in vectors within thecomposition to infect peripheral neurons associated with the sensationof pain. Within such neurons, the nucleic acid sequences encoding PP1αwithin the vectors are transcribed within the neurons to produce PP1αwithin the neurons. Typically, the mammal is human, but laboratoryanimal models (e.g., mice, rats, etc.), companion animals (e.g., cats,dogs, horses, etc.) or animals of zoological importance (apes and otherprimates, ungulates, antelopes, great cats, and other animals) or ofagricultural importance (cattle, goats, sheep, swine, etc.) can also betreated in accordance with the inventive method. The method can also beused to mitigate the sensation of pain caused by exposure to capsaicinor resiniferatoxin (RTX). Without wishing to be bound by any particulartheory, it is believed that the presence of PP1α within the neurons as aresult of the inventive method antagonizes the activity of TRPV1 withinthe neurons. It is believed that the method is specific to pain mediatedby TRPV1 (heat-related, capsaicin-induced, and associated with low pH),but does not mitigate pain associated with exposure to cold. Inparticular, it has been discovered that PP1α does not block TRPA1 orTRPM8 responses.

It will be recognized that, in carrying out the inventive methods,vectors other than those based on HSV can be used. For example, thevector can be derived from human or other mammalian adenoviruses,adeno-associated viruses, retroviruses, and the like. In someapplications, plasmids, liposomes, and other non-viral vector systemsalternatively can be employed. These vector systems, and their methodsof construction, propagation, and formulation for pharmaceutical use,are known to persons of ordinary skill.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the identification of a TRPV1 modulatory geneproduct from an HSV-based cDNA library screen for gene products thatblocks capsaicin-induced TRPV1 activation and cell death, allowing HSVvector plaque formation.

We sought to test the hypothesis of whether cellular modulators of TRPV1activation exist to control the appearance of long-term pain, a functionthat represents a viable target for down-regulation during thetransition from acute to chronic pain. To this goal, we employed anHSV-based cDNA library expression screen methodology based on a previousscreening method [Srinivasan et al., 2007] designed to identify cellularregulators of TRPV1 activation that may control the occurrence ofTRPV1-related nocifensive responses.

Our library screen was based on the observation that TRPV1 expression inVero cells, commonly used for HSV growth, caused rapid cell death viacalcium influx in the presence of capsaicin, suggesting that plaqueformation by an HSV-based TRPV1 expression vector in the presence ofcapsaicin would only occur if the vector also expressed an antagonist ofTRPV1 activation. To develop this system, we engineered a bacterialartificial chromosome (BAC)-based HSV-1 genome in E. coli to contain:(i) the cDNA for TRPV1 in place of the viral thymidine kinase (tk, UL23)gene and the UL41 virion host shut-off gene (vhs, UL41) under control ofthe HSV early (13) tk gene promoter; (ii) a Gateway (GW) recombinationcassette flanked by the hCMV1 major IE gene promoter and bovine growthhormone (bGH) polyA region in place of the internal repeat (joint)region of the virus; and (iii) a second antibiotic resistance gene(ampicillin-resistance) inserted between UL55 and UL56 (FIG. 1A); theregion between UL37 and UL38 contains a chloramphenicol resistance geneassociated with the BAC elements. The presence of both ampicillin andchloramphenicol during vector growth in E. coli prevents theamplification of defective recombinants that have lost either regionbetween the TRPV1 genes on the circular BAC genome (UL38-UL55 andUL56-UL37) due to recombination between the 2 copies of the TRPV1 gene.BAC DNA from thus selected E. coli was transfected into Vero cells andinfectious virus was harvested and titered as plaque forming units(pfu). In the presence of capsaicin, plaque formation was substantiallyreduced but not completely eliminated (1 plaque/10,000 input pfu), anacceptable background level for use in the cDNA library screen.

Next we recombined a previously described entry cDNA library [Wolfe etal., 2010] derived from a mixture of NGF-differentiated andundifferentiated PC12 cells into the GW locus of the double-TRPV1/amp(TTA) BAC and converted bulk recombinants into infectious virus (FIG.1B) with insert sizes ranging from 400 bp to ˜3 kb (FIG. 1B).Approximately 500,000 pfu of virus were screened in the presence of 1 mMcapsaicin (FIG. 1C) and a total of 46 candidate plaques were isolated,amplified on Vero cells and retested for capsaicin-resistance. Uponrescreening, approximately 50% of the isolated plaques retained acapsaicin-resistant phenotype. The cDNA inserts from candidates thatwere positive upon rescreening were then PCR amplified, introduced intoa new TTA BAC backbone and retested for capsaicin resistance to confirmthat the cDNA were not isolated from false negatives. One candidateclone consistently demonstrated the ability to rescue virus replicationin the presence of capsaicin and sequencing of this candidate insertidentified the gene as the full-length PP1α cDNA. Of note, this is thefirst observation within the literature in which TRPV1 activity isaltered by the expression of the PP1α phosphatase, although the PP2Bphosphatase calcineurin has been previously shown to have the ability todesensitize capsaicin-induced TRPV1 [Chaudhury et al., 2011; Docherty etal., 1996; Jeske et al., 2006; Mohapatra & Nau, 2005; Por et al., 2010].

We then tested the ability of the PP1α-TTA recombinant virus toreplicate in the presence of capsaicin with that of a positive controlvirus [Poreless (PL)-TTA] and a negative (TTA) control vector that isidentical to the PP1α-TTA except that it lacks any inserted gene. If thePP1α-TTA expressing PP1α acts to diminish TRPV1 activation in thepresence of the agonist capsaicin, then it will enable the virus to growto levels approaching that seen in the absence of capsaicin. This levelfor the PP1α-TTA recombinant should be similar to that seen with thePL-TTA positive control vector, since poreless TRPV1 acts in adominant-negative manner to inhibit TRPV1 activity [Srinivasan et al.,2007]. The negative control vector should not be able to reduce thecapsaicin induced sensitization of TRPV1, and thus one should seecalcium influx and cell death so that the TTA negative control can notreplicate. Vero cells were infected with low amounts of virus (MOI=0.01)and virus yield was measured 48 hours later. TTA virus produced noinfectious progeny while PP1α-TTA replicated with an efficiency similarto PL-TTA, both yielding approximately 3,000 pfu compared to 0.5-1×10⁶pfu produced in the absence of capsaicin (FIG. 2). The yields in theabsence of capsaicin were comparable between the three viruses,indicating that expression of PP1α and PL from the TTA vectors did notuniversally improve virus yield.

Having now confirmed that PP1α acts similarly to PL uponcapsaicin-induced sensitization of TRPV1, we tested of the function ofPP1α in neurons both in vitro and in vivo using a non-replicating HSVvector. Thus, we engineered 3 new vectors containing the strong hCMV IEpromoter driving PP1α, PL or eGFP cDNA inserted into the essential HSVICP27 IE gene locus in a vector backbone that was deleted for theessential ICP4 IE gene. These replication-defective vectors can only bepropagated on Vero cells engineered to complement the deleted ICP4 andICP27 essential IE genes in trans [Marconi et al., 1996], yet theyreadily express high-levels of the transgene in non-complementing cells,including neurons. As an in vitro test of the activity of the PP1αcandidate gene product to modify capsaicin-induced TRPV1 activity, wecompared the abilities of the replication-defective PP1α, PL and eGFPvectors to block the capsaicin-induced calcium influx in primary ratfetal DRG neurons in culture using Fura-2, a cell-permeable fluorescentCa²⁺ indicator [Zhang et al., 2011]. Capsaicin addition to the fetal DRGcultures should activate the opening of the TRPV1 channel, causing aninflux of Ca²⁺ into the neurons which will lead to a increase in Fura-2fluorescent signal, as observed for cultured DRGs that had been infectedwith the eGFP control virus (FIG. 3). However, if the candidate genereduces capsaicin-induced TRPV1 activity, one should observe a decreasein the Fura-2 fluorescent signal, as observed with the positive controlvirus expressing the PL protein that is known to interfere with TRPV1function following capsaicin treatment (FIG. 3). Finally, thePP1α-expressing vector, like the PL positive control vector, showedsignificantly reduced calcium uptake in the presence of capsaicincompared with the eGFP control vector (p=0.01).

The next goal was to determine whether PP1α expressed from the vectoralters nociceptor signaling via TRPV1 and if other TRP channels would beaffected as well by this phosphatase. Because TRPV1 has beenwell-documented to be involved in thermal pain responses such as thermalhyperalgesia (TH), we first tested the ability of the non-replicatingPP1α, PL (positive control) or eGFP (negative control) vectors toincrease paw withdrawal latency using a Hargreaves apparatus [Hargreaveset al., 1988; Goss et al., 2011]. Male Sprague-Dawley rats were injectedwith vector into the right footpad (n=6-8) and the TH nociceptiveresponses were recorded at 7-21 days with an average of three trials perhind paw. Each animal's thermal response was expressed as a ratiobetween the contralateral (non-injected) to ipsilateral (injected) paw,so that each rat acted as its own internal control. If thevector-expressed gene alters the TH response and the animal is notsensing the thermal-induced pain as readily, then the TH response forthe vector-injected ipsilateral paw increases in comparison to theun-injected paw and the TH response ratio will be >1. However, if thegene product has no effect on thermal-induced pain, then both paws willdisplay a similar response to heat and the response ratio will be ˜1.The positive control PL vector produced a 1.6-fold increase in pawwithdrawal latency compared to the eGFP control vector at 7 days postvector injection (FIGS. 4A-4B) and the PP1α-expressing vector yielded a1.4-1.7× increase in paw withdrawal compared to the eGFP control vectorover the period from 1-3 weeks post vector injection (FIGS. 4A-4B),again showing that the PP1α gene candidate acted in a manner similar tothe TRPV1 modulatory gene poreless (PL).

The next step was to confirm the role of PP1α in capsaicin-induceddesensitization of TRPV1 as we had observed in the screen itself as wellas in the tests of vector replication (FIG. 2) and calcium imaging (FIG.3) in response to the addition of the capsaicin TRPV1 agonist. To assessthis, we compared rats again injected into their right hindpaw with the3 vectors on a dynamic hot plate where temperature increased from 30° C.to 45° C. over a 15 minute period following injection of 10 mM capsaicinto the plantar surface of the injected ipsilateral paw. During eachdegree-minute interval, the total number of pain-related behaviors(licking, paw withdrawals) for the injected ipsilateral paw were countedand plotted. If the vector-expressed gene works via modulating TRPV1activity, it should display reduced numbers of nocifensive painfulresponses compared to the control vector, and this difference should beeven more pronounced at temperatures >42° C. since the TRPV1 channel ishighly activated by elevated temperatures, especially in the presence ofagonist. The PL positive-control vector reduced the number ofpain-related responses compared with the eGFP control vector as thetemperature of the heat ramp increased approaching 42° C. (FIGS. 5A-5B).While the differences were less significant at temperatures >42° C., thePP1α-expressing vector showed significant differences with the eGFPvector in the number of painful responses out to 45° C., attesting tothe ability of the PP1α-expressing vector to control the thermalnociceptive response that is specific for capsaicin-induced painsensitivity via TRPV1, confirming that PP1α is acting by modulating theactivity of TRPV1.

Since we had performed both in vitro and in vivo testing of the abilityof HSV vector-expressed PP1α to modify the activity of the TRP channelTRPV1 in models of thermal pain with and without capsaicin induction, wewished to test whether vector-expressed PP1α modulates other TRPreceptors such as TRPM8 and TRPA1. To assess whether PP1α can modulateTRPM8 activity, we performed a cold ramp assay that is identical to theheat ramp except that the rats injected into the right footpad with the3 vectors (GFP, PL, and PP1α; n=6 rats/vector group) are subjected totemperatures that decrease over the 15-minute period from 20° C. to 4°C. at a rate of 1° C. per minute. Again, like the heat ramp assay, thenumber of painful responses (withdrawal and licking of the ipsilateralhindpaw) was recorded at each temperature during the drop in cold ramptemperature. In this assay, neither the PL positive-control vector northe PP1α gene vector (FIGS. 6A-6B) displayed any difference from theeGFP negative-control vector.

We also performed the exact same cold ramp test following the injectionof either menthol or icilin into the plantar surface of thevector-injected ipsilateral footpad as was done with capsaicin in theheat ramp assay. Neither the PL nor PP1α vectors showed any differencesfrom the eGFP vector in the pain responses to injection of these TRPM8agonists in the context of decreasing temperatures from normal roomtemperature to 4° C. (data not shown). These negative resultsdemonstrate that PP1α does not modulate TRPM8 activity ordesensitization, verifying that it shows functional specificity for theTRPV1 TRP channel. A sole report exists in the literature that uses adrug inhibitor of PP1 (Okadaic Acid) to suggest that PP1 may be involvedin TRPM8 desensitization [Premkumar et al., 2005]. However, asacknowledged in the report, it is entirely possible that theconcentration of the inhibitor employed actually inhibited PP2A that isalso sensitive to the drug. Thus, this does not conflict with ourresults in the rat cold ramp model using either menthol or icilin asagonists of TRPM8.

Another TRP involved in sensation and the pain response is the TRPA1channel. To examine whether PP1α can modulate TRPA1 activity, we testedthe three vectors in the formalin footpad test where the late phaseinflammatory pain response to formalin injection into the hindpawinvolves GFRa+/IB4+ thinly myelinated fibers that express TRPA1 ratherthan TRPV1 [Akopian et al., 2007; Barabas et al., 2012; Salas et al.,2009]. As in all the in vivo studies mentioned above, the PP1α and eGFPcontrol vectors were injected into the right footpad of maleSprague-Dawley rats and the animals were tested after 7 days for theirnocifensive behavior following inoculation of a dilute solution offormalin into the ipsilateral vector-injected footpad. We determined theweighted pain score based on a series of pain responses by the rats[Dubuisson & Dennis, 1977] as in on our prior studies using HSV vectorsin the formalin model [Goss et al., 2001]. The data were plotted as theweighted pain score over a period of an hour following formalininjection (FIG. 7) and show the characteristic bi-phasic pain responsewhere the first response represents more acute spontaneous pain whilethe later response is that seen as a more chronic inflammatory painresponse previously shown to involve the TRPA1 receptor. PP1αvector-injected and eGFP control vector-injected animals showed minimaldifferences in their pain scores in response to formalin injection (FIG.7), showing that PP1α does not modulate the TRPA1 channel. While notexcluding effects on other pain-related channels, this observation isconsistent with a certain specificity of PP1α for responses involvingTRPV1.

In summary, using the creative cDNA library screening methodology, wehave now identified PP1α as a modulator of TRPV1, and shown that it isquite specific for pain behaviors linked to TRPV1 while PP1α has noeffect on TRPM8- or TRPA1-involved nocifensive responses. Moreover, wehave shown that replication-defective HSV-mediated delivery of PP1α canbe used to mitigate pain in a variety of animal models of pain thatarise via stimulation of the TRPV1 receptor, suggesting that it may makean effective therapeutic for some types of chronic pain.

Example 2

INTRODUCTION AND OBJECTIVES: Increased afferent excitability has beenproposed as an important pathophysiological basis of overactive bladder(OAB) and hypersensitive bladder disorders such as interstitialcystitis/bladder pain syndrome (IC/BPS). It has also been reported thattransient receptor potential TRPV1 receptors predominantly expressed inC-fiber afferent pathways greatly contribute to afferent sensitizationin these disease conditions. As shown in Example 1, HSV vector-mediatedPP1α expression leads to the reduction in capsaicin-induced thermalhyperalgesia. Therefore, this led us to investigate the effect of HSVvectors-mediated gene delivery of PP1α on TRPV1-mediated bladderoveractivity and pain-related behavior in rats.

METHODS: Replication-deficient HSV vectors encoding PP1α or greenfluorescent protein (GFP) as control were injected into the bladder wallof adult female Sprague-Dawley rats. Cystometry (CMG) under urethaneanesthesia was performed 1 week after viral injection to evaluatebladder overactivity induced by resiniferatoxin (RTX, a TRPV1 agonist).RTX-induced nociceptive behavior such as licking (lower abdominallicking) and freezing (motionless head-turning) was observed 2 weeksafter viral injection. Using immunohistochemistry, GFP expression inL6/S1 DRG and the bladder as well as c-Fos positive cells in the L6spinal cord dorsal horn were evaluated.

RESULTS: GFP expression was seen in L6/S1 DRG sections. In CMG, the PP1αgroup showed a significantly (p<0.05) smaller reduction (46.5±1.7%) inintercontraction intervals after RTX infusion than the GFP group(65.7±3.8%). The number of RTX-induced freezing behavior, which iscorrelated with bladder pain sensation, was significantly (p<0.001)decreased in PP1α vs. GFP groups. The number of c-Fos positive cells inthe L6 spinal dorsal horn was significantly (p<0.01) smaller in PP1α vs.GFP rats (24±4 vs. 59±6 cells per section).

CONCLUSIONS: These results indicate that HSV vectors injected into thebladder wall are transported to lumbosacral DRGs that contain bladderafferent neurons, and that PP1α gene delivery in the bladder iseffective in suppressing TRPV1-mediated bladder over-activity and painbehavior. Thus, HSV-mediated PP1α gene therapy could be effective forthe treatment of OAB and/or hypersensitive bladder disorders such asIC/BPS.

Example 3

This Example demonstrates the evaluation of the HSV-PP1α vector in a ratmodel of Post-Herpetic Neuralgia (PHN) pain.

We had previously established a rat model of Post-Herpetic Neuralgia(PHN) pain (Goins and Kinchington 2011 J. Neurovirol. 17(6):590-9; Garryet al., 2005 Pain 118(1-2):97-111) by injecting rat footpads with MeWocells infected with Varicella Zoster Virus (VZV) strain pOka, theparental of the Oka strain which is similar to the vaccine Oka strain(vOka). Since VZV is so cell-associated, one can only injectVZV-infected cells, not purified virus, the way one can do with HSV. Ourgroup and others have seen statistically significant changes in bothmechanical (MA) allodynia and thermal hyperalgesia (TH) nocifensivebehaviors in rats starting at 1-2 weeks post VZV injection lasting outto 5-9 weeks post VZV, a very robust model of chronic pain that mimicshuman patient PHN. Other groups have since employed this model toevaluate various drug treatment regimens to determine whether they haveany effects on VZV-induced PHN pain, with little to no effect. We haverecently tested the HSV-enkephalin vector in this model and have shownthat HSV vector-mediated enkephalin expression could block thenocifensive behaviors induced by VZV.

We have since initiated a study in which we compared the HSV vectorexpressing PP1α in the rat PHN model with vector expressing the GFPreporter gene. Baseline mechanical allodynia (MA) and thermalhyperalgesia (TH) nocifensive responses were measured in ten maleSprague Dawley rats using the von Frey hair up-down method (MA) or theHargreaves (TH) apparatus. Rats were then injected into their righthindpaws with MeWo infected with 10⁵ pfu VZV strain pOka (parent of Oka,similar to VZV vOka vaccine strain). At 7 and 14 days post VZV, MA andTH behaviors were measured. Next, on Day-0 the rats were injected intothe same hindpaws with 10⁷ pfu of either the control GFP-expressingreplication-defective HSV vector or the PP1α-expressing vector. AtDay-7, rat MA and TH behaviors were assessed. All behavioral scores wereplotted as the average response ratio for the ipsilateral injectedhindpaw to the contralateral un-injected hindpaw. VZV-PHN mechanical andthermal nocifensive behaviors were observed at Day 0. However, only thecontrol GFP-expressing animals retained these responses at Day 7 whilethe PP1α-expressing vector dramatically reduced both MA and THnocifensive behaviors in a statistically significant manner. The averagepaw withdrawal latency time for the uninjected non-inured rats rangedfrom 7-10 seconds while those for the VZV-injected/PP1α-vector injectedrats ranged from 13-25.

We initially injected VZV pOka into the footpads of 10 Sprague-Dawleyrats (Charles River) after measuring baseline MA and TH behaviors.Again, all behaviors are measured for the injected ipsilateral footpadcompared to the un-injected contralateral footpad, where if the rat isfeeling no pain the ratio will equal 1.0 while if the animal showsnocifensive behaviors the ratio will drop below 1.0 with the greater thedrop, the more indicative the magnitude of the painful response. Afterinjection of the VZV-infected MeWo cells into the ipsilateral ratfootpads, we again measured MA and TH behaviors and saw that for both MAand TH these responses approached scores around 0.5 or below (FIGS.8A-8B), indicative of pain. Next, the first group of 5 rats was injectedwith 10⁷ pfu of HSV control vector expressing GFP or the HSV-PP1αvector. Again, we measured MA and TH scores weekly following HSV vectorinjection into the same ipsilateral footpad. As seen in FIGS. 8A-8B,both the MA and TH scores at day 7 post HSV vector injection reversedthe painful responses to values of 1.0 or greater, and in the case of THbehaviors led to scores above 1.0, demonstrating that the HSV-PP1αvector worked to alleviate VZV-induced PHN pain.

We continued to follow the two groups of 5 rats further out, measuringboth MA and TH responses weekly following HSV vector injection. Baselinemechanical allodynia (MA) and thermal hyperalgesia (TH) nocifensiveresponses were measured in ten male Sprague Dawley rats using the vonFrey hair up-down method (MA) or the Hargreaves (TH) apparatus. Ratswere then injected into their right hindpaws with MeWo cells infectedwith 10⁵ pfu of VZV strain pOka. At 7 and 14 days post VZV, MA and THbehaviors were again measured. By 14 days post VZV injection, all ratsdisplayed painful behaviors assessed by MA and TH. Having establishedpain in these rats, on DAY-15 post VZV injection, the rats were injectedinto the same hindpaws with 10⁷ pfu of either the control GFP-expressingreplication-defective HSV vector or the PP1α-expressing rd HSV vector.At weekly intervals shown in the timeline at the bottom of FIG. 9C, ratMA and TH behaviors were assessed (FIGS. 9A-C). All behavioral scoresare plotted as the average response ratio for the ipsilateral injectedhindpaw to the contralateral un-injected hindpaw. VZV-PHN mechanical andthermal nocifensive behaviors were observed by 14 days post VZV pOka.However, only the control GFP-expressing animals retained theseresponses at Day 21 (7 days post HSV vector injection) while thePP1α-expressing vector dramatically reduced both MA and TH nocifensivebehaviors in a statistically significant manner. Even at 6 weeksfollowing HSV vector injection, the PP1α vector continues to block thepainful behaviors in the PHN rats at statistically significant levels.However, the extent of the response seems to be waning slightly in thethermal pain measurements at 5-6 weeks.

Thus, we have now tested the effects of HSV-PP1α vector mediatedexpression in a well established model of the chronic pain experiencedby VZV infected patients that does not respond well to any standard drugtherapies; patients either do not respond at all to drugs, or aftershort treatment regimens no longer obtain relief from those drugs,reinforcing the unmet need for an effective treatment for PHN pain.

Example 4

This Example demonstrates Action of HSV Vector-mediated PP1α in theComplete Freund's Adjuvant (CFA) Pain Model.

We have previously employed the complete Freund's adjuvant (CFA) modelto assess HSV gene therapy modalities such as the glycine receptor(GlyR) (Goss et al., 2011 Mol. Ther. 19(3):500-6) and endomorphin (Haoet al., 2009 Eur. J. Pain 13:38-6). In order to evaluate whether HSVvector mediated expression of PP1α can affect the inflammatory pain seenin rats injected into the footpad with CFA, 200-250 g maleSprague-Dawley rats (N=4/vector group) were injected subcutaneously intothe right rear hind-paw with 1×10⁸ pfu of either a control vector(DAP-GFP), a vector expressing PP1α (DAP-PP1α), or vector expressingPoreless-TRPV1 (DAP-PL). Three weeks later, thermal response was againassessed and the average and standard deviation of the thermal responsetimes (in seconds) shown for the injected right hind-paw as time point 0days post CFA injection. After this Day 0 measurement, 100 μL ofcomplete Freund's adjuvant (CFA) was injected into the right hind-paw aspreviously described (Goss et al., 2011) and the thermal response wasagain assessed at 1, 2 and 3 days post-CFA injection. Thermal pain wasassessed 1-4 days post CFA where the inflammatory response is greatestby 24-48 h, but begin to abate and is back to normal or near that by 4-5days. We noticed that the CFA-injected hind paws were red and inflamedat 24 h, decreased somewhat on day 2, even less so by day 3 and nearnormal except for some residual scarring on day 4.

Although all the rats displayed TH latency withdrawal times within therange of 6-7 seconds prior to injection of any of the three vectors(DAP-GFP, DAP-PL, and DAP-PP1α), only the rats injected with the DAP-GFPcontrol vector demonstrated a similar latency withdrawal time at 3 weekspost-infection (FIG. 10 Day 0). Rats in the DAP-PL, and DAP-PP1α groups,showed increased withdrawal times between 7.6-8.4 seconds, as we hadseen in previous thermal pain analyses demonstrating that the geneproducts expressed by these vectors alter the thermal pain response.Once the rats were, the DAP-GFP control rat group TH latency withdrawaltimes dropped from 6 to 2.5 seconds within the first 24 hours after CFAinjection. The DAP-PL rats dropped only slightly from 8.4 to 5.9seconds, while the DAP-GFP group dropped from 7.6 to 7.0 seconds. Eventhough the DAP-GFP group continued to respond to the thermal source inslightly over 2 seconds at 48 h, the DAP-PL, and DAP-PP1α group thermaltimes each increased by around 1 second, and basically improved tolevels observed at time 0 prior to CFA injection. These results showthat like porless-TRPV1, vector-expressed PP1α altered the rats' thermalresponse during the inflammatory reaction caused by the injection of CFAin a manner similar to which we have previously seen using vectors thatexpress either endomorphin (Hao et al., 2009 Eur. J. Pain 13:38-6) andthe glycine receptor (GlyR) (Goss et al., 2011 Mol. Ther. 19(3):500-6).These results support the conclusion that PP1α can ameliorateinflammatory nocifensive behaviors.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein. he following references also are expressly incorporated byreference:

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The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-22. (canceled)
 23. A herpes simplex virus (HSV) vector comprising anexpression cassette, wherein the expression cassette comprises a nucleicacid sequence encoding PP1α operably linked to a non-native PP1αpromoter, and wherein the expression cassette is inserted into the ICP27locus of the HSV vector.
 24. The HSV vector of claim 23, wherein thenucleic acid sequence encodes the human form of PP1α.
 25. The HSV vectorof claim 23, wherein the non-native promoter is a strong constitutivepromoter.
 26. The HSV vector of claim 25, wherein the strongconstitutive promoter is the hCMV1 promoter.
 27. The HSV vector of claim23, wherein the non-native promoter is a non-neuronal promoter thatprovides long-lasting gene expression within neurons.
 28. The HSV vectorof claim 27, wherein the non-native promoter is a ubiquitin (Ub)promoter.
 29. The HSV vector of claim 23, wherein the non-nativepromoter is a neuronal-specific promoter.
 30. The HSV vector of claim23, wherein the non-native promoter is the transient receptor potentialcation channel subfamily V member 1 (TRPV1) promoter.
 31. The HSV vectorof claim 23, which lacks functioning genes encoding ICP4 and ICP27. 32.The HSV vector of claim 31, further comprising at least one othermutation.
 33. The HSV vector of claim 23, which is targeted tospecifically infect C-fibers within a mammal.
 34. The HSV vector ofclaim 33, wherein the mammal is human.
 35. A viral stock comprising adefined titer of the HSV vector of claim
 23. 36. A pharmaceuticalcomposition comprising the HSV vector of claim 23 and apharmaceutically-acceptable carrier.
 37. The pharmaceutical compositionof claim 36, formulated as a liquid suitable for administration by skinprick, or via subdermal, intramuscular, or parenteral injection.
 38. Thepharmaceutical composition of claim 36, formulated for transdermaladministration.
 39. A method of treating pain within a mammal in needthereof, comprising administering an effective amount of thepharmaceutical composition of claim 36 to the mammal such that PP1α isproduced within peripheral neurons associated with the sensation ofpain.
 40. The method of claim 39, wherein the pain is caused by exposureto capsaicin or Resiniferatoxin (RTx).
 41. The method of claim 39,wherein the mammal is human.